Two new classes of inhibitors of LpPLA2 have been identified in fermentations of Pseudomonas fluorescens. The two structurally isomeric series differ in the geometry of closure of the bicyclic carbamate and comprise a range of compounds varying only in length of their lipophilic sidechain. The most abundant species were extracted from the cells and purified by silica and C18 chromatography. Members of the more stable class were shown to be potent and selective competitive inhibitors of LpPLA2.

Fermentation development for improved culture productivity can be achieved in a number of ways. Conventional approaches usually concentrate initially on optimisation of the final stage fermentation. However an understanding of the seed stage and its further development can lead to an improvement in final stage productivity. A significant increase in the production of milbemycin VM44866 byStreptomyces hygroscopicus was achieved by manipulation of several factors associated with the seed stage fermentation. Juvenile seeds and seed media containing reduced levels of carbohydrates overcame the detrimental effects of passaging and seed age associated with the standard (control) process. The effect of final stage inoculum level was seed medium-dependent and seed fermentation incubation temperature also affected subsequent milbemycin VM44866 production. These findings were extended to a second milbemycin-producing strain and these results have demonstrated the potential benefits of seed stage optimisation for improved final stage production.

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Milbemycin production byStreptomyces hygroscopicus RB4569D was examined in media containing different carbohydrates. Total milbemycin titer could be increased by substitution of fructose for glucose and by selection of the appropriate starch type. Total titer could be further enhanced by increasing the concentration of fructose and/or starch in the medium. Rates of carbohydrate utilization were shown to be independent of their initial concentration and increased titers in high carbohydrate media were shown to be due to a prolonged production phase rather than an increased accretion rate. The pattern of individual milbemycin components was governed by the carbon:nitrogen ratio of the medium rather than carbohydrate concentration and there was a critical C:N ratio below which no milbemycin was produced.

Abstract. The solutes accumulated by cyanobacteria in response to hyper‐osmotic stress include Na+, K+, sucrose, trehalose, glucosyl‐glycerol, glyeine betaine and glutamate betaine. The compatibility of several of these solutes with glutamine synthetase activity has been examined using cell‐free extracts from a range of freshwater, marine and halotolerant cyanobacteria. All of the solutes tested were compatible with (i.e. non‐inhibitory to) enzymic activity at physiological concentrations and the results demonstrate a rank order of compatibility which correlates with the concentrations at which the organic solutes occur in cyanobacteria, i.e. glycine betaine > polyol‐derivatives > disaccharides and with the upper salinity limit for growth. The protection against inhibition by NaCl (halo‐protection) afforded by these solutes to enzymic activity was also examined. Only glycine betaine was found to exert a significant halo‐protective effect and this may be explained by differences in the mechanism of compatible solute function between small charged molecules and sugars/polyols.

Rates of accumulation of low-molecular-weight carbohydrates have been measured for a range of cyanobacteria subjected to hyperosmotic stress (upshock). Synthesis of the disaccharides sucrose and trehalose was more rapid than that of the heteroside glucosyl-glycerol in all strains. The rates of synthesis of organic solutes may affect the distribution of cyanobacteria in natural saline waters:strains that produce either sucrose or trehalose in response to increased external salinity are often found in brackish habitats where the salinity regime is unstable and rapid organic solute synthesis may be advantageous. In contrast, cyanobacteria that synthesize glucosyl-glycerol are more commonly isolated from environments of less variable salinity.

Three groups of cyanobacteria are recognized on the basis of their organic osmotica and upper salinity limit for growth. In general, the least halotolerant forms accumulate disaccharides, while cyanobacteria of intermediate halotolerance synthesize the heteroside glucosylglycerol and the most halotolerant isolates accumulate betaines in response to salt stress. However, certain strains also accumulate additional organic solutes, depending upon the growth temperature, the ambient salinity and the duration of salt stress.

A procedure is described for the release of low molecular weight carbohydrates and free amino acids from the unicellular cyanobacteria Synechocystis PCC6714 and Synechococcus PCC6311. Metabolite release was induced by rapid transfer of free-living or immobilized cells grown in a medium containing 490 mol m−3NaCl to a freshwater-based medium (BG-11); up to 50% of the low molecular weight carbohydrates and 73% of the intracellular free amino acids were released within 2 min. Cells of Synechocystis PCC6714 were subjected to repeated osmotic shock, with no evidence of long-term damage to the cells after three cycles of metabolite release over a 48 h period. Because this procedure does not involve harvesting or death of the cells, it may prove to be a useful means of recovering metabolites from viable microorganisms.

Photosynthetic, prokaryotic blue-green algae (cyanobacteria) occur in a wide range of natural habitats of diverse ionic composition and as such, represent an important source of biological material for biosolar energy conversion programs using saline water. The gasvacuolate, filamentous Spirulina is grown in ‘seminatural’ culture in Lake Texcoco, Mexico, as a major source of single-cell protein for animal nutrition. Pilot-scale trials in other areas of the world have also demonstrated the suitability of blue-green algae, including Spirulina, for growth under brackish conditions. The carbohydrate accumulation profiles of blue-green algae differ in isolates from freshwater, marine and hypersaline habitats, with a trend towards sucrose or trehalose accumulation in stenohaline freshwater strains grown in media containing NaCl, while euryhaline and marine forms frequently accumulate glucosylglycerol. Many halotolerant isolates from hypersaline habitats accumulate glycinebetaine in response to osmotic stress. This knowledge may provide scope for future improvement in the N2 fixation rates of blue-green algae in saline media, using betaine-accumulating N2-fixing strains in preference to other, saltsensitive isolates.

Transfer of Synechocystis PCC6714 from a freshwater medium to a saline medium caused the cells to shrink; rapid entry of NaCl resulted in a partial recovery of cellular volume within 2 min. Active extrusion of internal Na+ in exchange for extracellular K+ then occurred (within 20 min). Finally, the low-Mr carbohydrates sucrose and glucosylglycerol were accumulated and internal KC1 levels declined. In long-term growth experiments, the relative importance of sucrose as a component of the low-Mr organic solute fraction decreased and glucosylglycerol became the single most important intracellular solute. These observations demonstrate that several inorganic and organic solutes are involved in osmotic adjustment in this cyanobacterium, with sequential changes in the relative importance of each solute following transfer to a saline medium.

SummaryThe effects of temperature and salinity on the carbohydrate accumulation profiles of a range of unicellular cyanobacteria (blue‐green algae) have been studied. Four isolates of Synechocystis sp. which usually accumulated glucosyl‐glycerol produced a second carbohydrate, sucrose, when grown in a seawater‐based medium at high temperature (35°C). In contrast, three sucrose‐accumulating isolates of Synechococcus, grown over a range of temperatures and salinities, did not produce a second detectable low molecular weight carbohydrate in any experimental treatment. The interaction of temperature and salinity was further examined in Synechocystis PCC 6714. Sucrose production was found to be favoured by high temperature and low salinity. The rate and extent of sucrose accumulation differed in cells grown at 20 and 37 °C reaching an upper asymptote 12 h after upshock at 37 °C and 24 h after upshock at 20 °C followed by a decrease to an approximately constant level after 96 h. This is the first demonstration of the accumulation of sucrose as a second osmotically active carbohydrate in cyanobacteria.

The filamentous cyanobacterium Spirulina platensis has been examined for salt tolerance and osmotic adjustment. Salinities up to 150% seawater had little effect on growth yield or photosynthetic O2 evolution; higher salinities were markedly inhibitory. Osmotic adjustment was achieved by the intracellular accumulation of the low-molecular-weight carbohydrate glucosyl-glycerol in response to increased external salinity: in fullstrength (100%) seawater glucosyl-glycerol accounted for approximately 5.0% of the dry weight of the cyanobacterium. Trehalose was also present, particularly in cells at low salt concentration, and in 50% seawater medium accounted for up to 1.0% of the dry weight of the cyanobacterium. For cells grown in 100% seawater the ratio of trehalose to glucosyl-glycerol varied with temperature: at 37°C trehalose comprised 31% (w/w) of the low-molecular-weight carbohydrates while at 20°C only 9% of the total was trehalose. When subjected to hypo-osmotic shock the intracellular concentration of glucosyl-glycerol decreased and this was mirrored by an increase in glycogen. An understanding of the osmotic adjustment of S. platensis has implications both for the mass culturing of this and other strains of Spirulina and possibly also for the quality of the harvested product.

The effects of NaCl, KCl and the organic osmotica sucrose or glycine betaine on glutamine synthetase (GS) transferase activity in crude extracts of the marine Nodularia harveyana and the halotolerant Synechocystis sp. DUN52, respectively, were examined. NaCl at low concentrations (< 0.75 M) stimulated the enzyme (up to 30%) in the halotolerant strain but was inhibitory above 0.2 M in the marine strain. In both strains KCl (0.1-1.3 M) stimulated GS activity and was inhibitory only above 1.4 M. Sucrose, the major intracellular organic osmoticum in N. harveyana, had only a slight inhibitory effect until concentrations above 0.3 M; 2.0 M sucrose caused a 60% inhibition. The quaternary ammonium compound glycine betaine, the primary organic osmoticum in Synechocystis sp. DUN52, was not inhibitory at physiologically relevant concentrations up to 1.8-2.0 M and reduced NaCl inhibition by 10-30% at NaCl concentrations from 0.8 to 2.0 M in the halotolerant strain when used at 1.0 M. At NaCl concentrations above 0.2 M, 0.2 M KCl reduced the activity of GS by 6-15%. Sucrose and KCl showed similar degrees of protection against NaCl inhibition in N. harveyana when used at concentrations similar to those measured in seawater-grown cells of N. harveyana. Increasing concentrations of glycine betaine increased protection of GS activity against NaCl inhibition at NaCl concentrations above 0.5 M. These results suggest that the extent of salt tolerance of a particular strain of cyanobacterium may depend, in part at least, on the metabolic effects of compounds that are accumulated as internal osmotica in that strain.

The effects of salinity stress on biomass yield, photosynthetic O2 evolution and nitrogenase activity were investigated using axenic cultures of Nodularia harveyana (Thwaites) Thuret originally isolated from a salt marsh at Gibraltar Point, Lincolnshire, UK in 1971 and studied in this laboratory in 1983. Biomass yields, as chlorophyll a per culture, were highest in the 0 to 100% seawater (0 to 35‰ sea salt) range with negligible growth in 200% seawater; growth on NH 4 + was greater than on N2 and NO 3 - , which did not differ significantly from each other. In short-term experiments, photosynthetic O2 evolution remained high at salinities up to 150% seawater (52.5‰ sea salt); nitrogenase activity remained high at salinities up to 100% seawater (35‰ sea salt). The major internal low molecular weight carbohydrate which accumulated in response to increased salinity was sucrose, the levels of which fluctuated markedly and rapidly in response to salinity change.

In a comprehensive survey of the carbohydrate accumulation profiles of more than 70 strains of cyanobacteria three organic osmotica (glucosylglycerol, sucrose and trehalose) have been identified in both freshwater and marine isolates under conditions of osmotic stress. While the trend was towards glucosylglycerol accumulation in marine strains and sucrose accumulation in freshwater forms, there were no absolute differences between cyanobacteria isolated from each habitat. There was also no clear link between genus and the type of carbohydrate accumulated.